Enteropathogenic E. coli (EPEC) are an important cause of diarrhea in infants. The long-term objectives of this project are to understand the pathogenesis of disease due to this organism and to develop diagnostic reagents and vaccine candidates for prevention of disease due of disease. There are also many similarities between the pathogenesis of EPEC and the pathogenesis of E. coli 0157:H7, the enterohemorrhagic E. coli (EHEC) which have been responsible for recent large outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS) in the U.S. due to ingestion of contaminated beef, water, apple cider and other vehicles. We have shown that there is genetic similarity between some virulence factors of EPEC and EHEC and information resulting from the proposed studies will also yield insights into the pathogenesis of EHEC. We have proposed a three stage model of EPEC pathogenesis and identified genes for virulence factors of each stage. The first stage consists of non-intimate adherence of the bacteria to epithelial cells which is mediated by a type IV pilus, the Bundle-Forming Pilus (BFP). The second stage involves a signal transduction event between the bacteria and the epithelial cell resulting marked cytoskeletal changes, dissolution of the microvilli, increase in intracellular calcium, and tyrosine phosphorylation of a 90 kDa epithelial cell protein. The third stage involves an intimate attachment between the bacteria and the epithelial cell with a concentration of polymerized actin and other cellular components accumulating at the site of bacterial attachment. There are three broad aims for the proposed experiments: l) Mutagenize and characterize a large (ca. 35 kb) region of the chromosome which contains multiple genes encoding virulence factors of EPEC. 2) Determine the extent of a putative virulence regulon in which the transcriptional activator PerA regulates a variety of EPEC virulence genes. 3) Characterize the effect of EPEC on intestinal epithelial cells using polarized intestinal cell lines and rabbit models. The proposed experimental approach will use a combination of molecular genetics, cell biology, and animal studies to achieve a full understanding of how EPEC infects epithelial cells and effects the ion secretion which presumably is responsible for diarrhea caused by EPEC.